Park Ji Yeong, Jeong Seon-Ju, Kim Jeong Hwan
Division of Applied Life Science (BK21 Plus), Graduate School, Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, 660-701, Republic of Korea.
Biotechnol Lett. 2014 Sep;36(9):1791-9. doi: 10.1007/s10529-014-1539-9. Epub 2014 Apr 26.
Lactic acid bacteria (LAB) were isolated from Kimchi, a Korean traditional fermented vegetable food. LAB accumulating GABA (γ-aminobutyric acid) in the culture media were screened by TLC analysis. One isolate, GU240, produced the highest amount of GABA among the 3,000 isolates and identified as a Lactobacillus zymae strain. Glutamate decarboxylase (GAD) gene was cloned and over-expressed in E. coli BL21(DE3) using pET26b(+). The recombinant GAD was purified by using a Ni-NTA column. Its size was 53 kDa by SDS-PAGE. Maximum GAD activity was at pH 4.5 and 41 °C and the activity was dependent on pyridoxal 5'-phosphate. Km and Vmax of LzGAD were 1.7 mM and 0.01 mM/min, respectively, when glutamate was used as a substrate.
乳酸菌（LAB）是从韩国传统发酵蔬菜食品泡菜中分离出来的。通过薄层色谱分析筛选出在培养基中积累γ-氨基丁酸（GABA）的乳酸菌。在3000株分离菌中，一株名为GU240的分离菌产生的GABA量最高，并被鉴定为发酵乳杆菌菌株。利用pET26b(+)载体在大肠杆菌BL21(DE3)中克隆并过量表达谷氨酸脱羧酶（GAD）基因。重组GAD通过镍-亚氨基二乙酸（Ni-NTA）柱进行纯化。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定其大小为53 kDa。GAD的最大活性在pH 4.5和41℃，且该活性依赖于磷酸吡哆醛。当以谷氨酸为底物时，发酵乳杆菌GAD（LzGAD）的米氏常数（Km）和最大反应速度（Vmax）分别为1.7 mM和0.01 mM/分钟。
